PROJECT SUMMARY Diabetic kidney disease (DKD) is a major debilitating complication of diabetes and a significant healthcare problem. Current therapies for DKD are not fully efficacious, with many patients still progressing to renal failure. Inadequate understanding of mechanisms involved in DKD has restricted the development of effective therapies, which underscores the critical need to identify novel mediators and mechanisms leading to DKD. In the previous funding period, we made a significant impact by demonstrating the involvement of non-coding RNAs (ncRNAs), including microRNAs (miRNAs) and long ncRNAs (lncRNAs), in the pathogenesis of DKD. We characterized a novel lncRNA, lncMGC, in mesangial cells, which is a host transcript of a mega cluster of miRNAs, the miR-379 cluster. We found that lncMGC modulates critical factors associated with DKD and that a GapmeR antisense oligonucleotide targeting lncMGC ameliorates pathological features of early DKD. However, the molecular and epigenetic mechanisms by which lncMGC promotes features of DKD, the effects of lncMGC, or miR-379 deficiency in vivo on diabetes/obesity-induced renal dysfunction in mice, and sex-specific effects are unclear. The current renewal will address these crucial gaps in knowledge using innovative technologies and mice. The objective of this proposal is to characterize the role of downstream targets and epigenetic regulators of the IncMGC-miR-379 axis in processes that lead to renal dysfunction and DKD. Extensive new preliminary data supporting our research goals include identification of: a) key protein binding partners of lncMGC associated with chromatin remodeling; b) new targets of miR-379 related to DKD pathology; and c) development highly innovative mouse models by CRISPR-Cas9 editing. We hypothesize that diabetes re-programs the mesangial cell (MC) transcriptome and epigenome to dysregulate lncMGC, hosted miR-379, and their key target genes that affect mitochondrial function, oxidative and ER stress resulting in fibrosis, renal dysfunction, and DKD. Specific Aim 1 will use state-of-the-art Omics profiling and technologies in MCs to determine novel protein targets and epigenetic mechanisms by which the lncMGC-miR-379 axis promotes DKD. Specific Aim 2 will use novel mouse models of miR-379 and lncMGC deficiency to evaluate the in vivo roles of lncMGC, miR-379, and related critical targets in DKD. Specific Aim 3 will utilize a novel humanized lncMGC mouse and GapmeR targeting human lncMGC to evaluate the translational potential for human DKD treatment. Because ncRNAs have essential roles in disease states, these continuing studies are both scientifically and clinically significant for DKD research. The project is innovative because it uses cutting edge technologies, novel mouse models, and translational methods. Together, they can alter existing paradigms and have a positive impact by uncovering new ncRNA-mediated regulation of DKD progression, with potentially far-reaching clinical and therapeutic implications, particularly for patients not responding to currently available treatments.